CD4+CD25+Foxp3+ regulatory T cells (Treg) have emerged as one of the key elements important for establishing and maintaining tolerance. While Treg have been the focus of much research, aspects of their basic physiology and function remain incompletely understood. In particular, it is not known where and when Treg or their precursors precisely interact with antigen, become activated, and deploy their suppressive regulatory mechanisms. The cellular and molecular mechanisms important for each of these steps are poorly delineated, and the migration and trafficking mechanisms essential for priming at one site and effector function in another site remain largely unknown. In the previous funding period, we published a number of important studies revealing that naive T cells migrate to lymph nodes (LN), where they are stimulated in the cortical area by specific alloantigen presenting plasmacytoid dendritic cells (pDC) to generate adaptive or induced Treg (aTreg). We elucidated several important molecular mechanisms in these interactions. In ongoing studies, we have made several additional key observations. First, while investigating the cell surface receptor signals and transcriptional factors that regulate Foxp3 expression, we have discovered that we can manipulate epigenetic regulation of the Foxp3 gene, through DNA methyltransferase (DNMT) inhibitors, to generate Treg. Second, in an islet transplant model we have discovered that nTreg migrate sequentially from the blood through microvascular endothelium into the site of tissue inflammation, and then subsequently from tissue into afferent lymphatics and then the draining LN (dLN), in order to be activated and fully display their suppressive functions. Sequential tissue and dLN migration are key components of nTreg physiology, and migration is coupled to developmental and differentiative steps. In contrast, aTreg precursors migrate first to the LN, and then to sites of inflammation. Third, Treg specifically express increased levels of lymphotoxin-? (LT?) on their cell surface; while endothelial cells, particularly lymphatic vascular endothelial cells, express increased levels of LT? receptor (LT?R). Inhibiting the LT?1?2-LT?R interaction inhibits Treg translymphatic migration, and inhibits tolerance to islet and cardiac allografts. Together these observations suggest that aTreg and nTreg have distinct requirements for migration, trafficking and suppressive function, and that these distinctions are critically important for immune suppression and regulation.